ABSTRACT
Maculatin 1.1 (Mac1) showed potent activity against Staphylococcus aureus with an MIC of 7 µM. The mode of action of Mac1 was investigated by combining assays with S. aureus cells and lipid vesicles mimicking their membrane composition. A change in Mac1 conformation was monitored by circular dichroism from random coil to ca. 70% α-helix structure in contact with vesicles. Electron micrographs of S. aureus incubated with Mac1 showed rough and rippled cell surfaces. An uptake of 65% of small (FD, 4 kDa [FD-4]) and 35% of large (RD, 40 kDa [RD-40]) fluorescent dextrans by S. aureus was observed by flow cytometry and indicate that Mac1 formed a pore of finite size. In model membranes with both dyes encapsulated together, the full release of FD-4 occurred, but only 40% of RD-40 was reached, supporting the flow cytometry results, and indicating a pore size between 1.4 and 4.5 nm. Finally, solid-state nuclear magnetic resonance showed formation of an isotropic phase signifying highly mobile lipids such as encountered in a toroidal pore structure. Overall, Mac1 is a promising antimicrobial peptide with the potent capacity to form pores in S. aureus membranes.
Subject(s)
Amphibian Proteins/pharmacology , Anti-Bacterial Agents/pharmacology , Antimicrobial Cationic Peptides/pharmacology , Cell Membrane Permeability , Cell Membrane/drug effects , Staphylococcus aureus/drug effects , Amphibian Proteins/chemical synthesis , Anti-Bacterial Agents/chemical synthesis , Antimicrobial Cationic Peptides/chemical synthesis , Cell Membrane/metabolism , Circular Dichroism , Dextrans/pharmacology , Drug Evaluation, Preclinical , Fluorescence , Lipid Bilayers/metabolism , Microscopy, Electron, Scanning , Molecular Weight , Porosity , Protein Structure, Secondary , Staphylococcus aureus/metabolism , Staphylococcus aureus/ultrastructureABSTRACT
We present a novel NMR-based study of the molecular aspects of the "attack" on human red blood cells (RBCs) by growing bacteria. Staphylococcus aureus expresses virulence factors, including alpha-hemolysin, which contribute to the clinical condition known as septic shock. alpha-Hemolysin is a pore-forming toxin and its secretion increases the permeability of a range of mammalian cell types infected with S. aureus. (31)P NMR spectra of the probe molecules dimethyl methylphosphonate (DMMP) and hypophosphite (HPA) in RBC suspensions show separate intra- and extracellular resonances. These resonances coalesced over time in RBC suspensions inoculated with S. aureus or pure alpha-hemolysin, due to increasing permeability of the RBC membrane. Increased RBC permeability resulted in leakage of intracellular proteins, plus an increase in the exchange rate of the solutes between the intra- and extracellular compartments, both effects contributing to the coalescence of the split peaks. The addition of antibiotics prevented peak coalescence and enabled the minimal inhibitory concentration (MIC) for eight strains of S. aureus to be determined for oxacillin and erythromycin. The MIC values obtained by using (31)P NMR spectroscopy were within one dilution of the MICs obtained using the standard National Committee for Clinical Laboratory Standards (NCCLS) method. The results are encouraging for the use of NMR spectroscopy in clinical microbiology.
Subject(s)
Magnetic Resonance Spectroscopy/methods , Sepsis/diagnosis , Staphylococcal Infections/diagnosis , Anti-Bacterial Agents/pharmacology , Bacterial Toxins/analysis , Erythrocytes/chemistry , Erythrocytes/microbiology , Hemolysin Proteins/analysis , Humans , Microscopy, Interference , Organophosphorus Compounds , Permeability , Phosphinic AcidsABSTRACT
Invasive aspergillosis remains a potentially life-threatening infection, the incidence of which is increasing. Current methods used to determine the susceptibilities of Aspergillus strains to antifungal drugs are often unreliable. Nuclear magnetic resonance (NMR) spectroscopy can identify the metabolic complement of microorganisms while monitoring nutrient utilization from the incubation medium. We used 600-MHz (1)H NMR spectroscopy to monitor the metabolic responses of five Aspergillus species cultured in RPMI 1640-2% glucose-morpholinepropanesulfonate buffer to various concentrations of the antifungal drugs amphotericin B (AMB) and caspofungin. The metabolic endpoint (MEP) was determined from nutrient and metabolite resonances, measured as a function of the drug concentration, and was defined as a > or =50% reduction in nutrient consumption or metabolite production. MICs were evaluated by a modification of Clinical and Laboratory Standards Institute broth microdilution method M27-A, and minimal effective concentrations (MECs) were determined by microscopic examination of fungal hyphae. For AMB, the MEPs coincided with the MICs. For caspofungin, the MEPs agreed with the MECs for several Aspergillus strains, but the effect of drug pressure was more complex for others. Expansion of the MEP definition to include any significant changes in metabolite production resulted in agreement with the MEC in most cases. Paradoxical metabolic responses were observed for several Aspergillus strains at either high or low caspofungin concentrations and for one Aspergillus terreus strain with AMB. NMR spectroscopy proved to be a powerful tool for detecting the subtle effects of drug pressure on fungal metabolism and has the potential to provide an alternative method for determining the susceptibilities of Aspergillus species to antifungal drugs.
